1. Technical Field
The present invention relates, in general, to a method of targeting DNA and, in particular, to a method of effecting sequence-specific targeting of DNA.
2. Background Information
Several groups have recently reported highly efficient cleavage of genomic DNA at specific sequences. For example, Szybalski and coworkers have cleaved Saccharomyces cerevisiae and E. coli genomes at a single introduced lac operator site (Koob et al, Science 250, 271 (1990)). These investigators first methylated Hae II sites in the DNA while using the lac repressor to protect the lac operator from methylation. After inactivation of the methylase and the repressor, the only Hae II site unmodified and available for cleavage was the lac operator site. The advantages of this approach were the high yield and high specificity. The disadvantage was that only a lac operator site could be cleaved.
A second approach was used by several investigators to cleave genomes as large as S. cerevisiae. This approach used the ability of synthetic homopyrimidine oligonucleotides to anneal to duplex homopyrimidine-homopurine tracts to form triple-helical structures. This approach was first used by Moser and Dervan (Science 238, 645 (1987)) to cleave a plasmid by equipping the oligonucleotide with an EDTA-Fe cleavage moiety. Subsequently, other cleavage moieties were attached to homopyrimidine oligonucleotides. For example, Schultz and coworkers attached staphylococcal nuclease (Pei et al, Proc. Natl. Acad. Sci. USA 87, 9858 (1990)), and Helene and coworkers attached a phenanthroline-copper derivative (Francois et al, Proc. Natl. Acad. Sci. USA 86, 9702 (1989)) as cleavage moieties. Dervan and coworkers have also used a guanine-rich cleavage oligonucleotide to form a triplex (Beal et al, Science 251, 1360 (1991)), and have cleaved DNA using a triplex and the methylation protection strategy described above (Strobel et al, Nature 350, 172 (1991)). The advantages of this targeting approach are efficiency and the ability to use oligonucleotides with a variety of derivatives. The disadvantage is that only homopyrimidine or guanine-rich oligonucleotides have been used successfully.
The practical use of previously reported strategies is severely limited because of the paucity, or indeed the complete absence, of possible cleavage sites in any particular DNA sequence. The present invention, on the other hand, provides a general method of cleaving DNA at any desired site, or pair of sites. Site-specific cleavage, however, is only a single embodiment of the present invention. In a broader sense, the invention relates to a method of targeting any desired sequence specifically and efficiently whether, it be for purposes of cleavage, protection or enrichment.